Tree Genetics and Molecular Breeding 2025, Vol.15, No.5, 192-201 http://genbreedpublisher.com/index.php/tgmb 193 cacao seeds. Their synthetic starting points are nucleotides such as adenosine monophosphate (AMP) and guanosine monophosphate (GMP). After a series of transformations, it is first converted into xanthosine, and then different methyl xanthine compounds are formed through methylation reactions. Theobromine is the most abundant purine alkaloid in the cacao tree, especially accumulating in large quantities in young tissues and the later stage of seed development (Koyama et al., 2003; Zheng et al., 2004; Pereira-Caro et al., 2013). 2.2 Enzymatic steps from xanthosine to theobromine (XMT, MXMT, DXMT enzymes) The synthesis process of theobromine mainly consists of several steps. Firstly, xanthosine is converted into 7-methylxanthosine under the action of 7-methylxanthosine methyltransferase (XMT). Then, 7-methylxanthine nucleoside hydrolyzes to form 7-methylxanthine. Then, under the action of 3-methylxanthine methyltransferase (MXMT), it is further methylated to form theobromine (3,7-dimethylxanthine). In some tissues, theobromine can still be converted into caffeine (1,3,7-trimethylxanthine) under the action of 1-methylxanthine methyltransferase, but this process is relatively slow in Theobroma cacao, so the content of theobromine is much higher than that of caffeine (Koyama et al., 2003; Pereira-Caro et al., 2013). 2.3 Regulation of metabolic flux in caffeine and theobromine pathways The accumulation of theobromine and caffeine can be influenced by tissue type, developmental stage and enzyme activity. Studies have found that theobromine has the highest content in young fruits and leaves, gradually decreases with maturity, but increases significantly in the later stage of seed development (Zheng et al., 2004; Pereira-Caro et al., 2013). The synthesis rate of caffeine is relatively low, mainly limited by the activity of the DXMT enzyme. Therefore, theobromine is the main end product in Theobroma cacao. In addition, metabolic fluxes are also regulated by gene expression and substrate specificity. The substrate specificity of N-methyltransferase determines the relative ratio of theobromine to caffeine (Koyama et al., 2003; González et al., 2019). Genomic studies have also shown that theobromine metabolism-related genes were subjected to selective pressure during domestication, which affected the accumulation pattern of alkaloids in different varieties (Cornejo et al., 2018). 3 Metabolomic Profiling of Theobromine in Cacao 3.1 Analytical platforms: LC- MS, GC- MS, and NMR for alkaloid profiling Theobromine is the main methyl xanthine alkaloid in Theobroma cacao. Metabolomics analysis of it relies on multiple highly sensitive platforms. Common methods include high performance liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), which can effectively separate and detect methyl xanthine compounds in complex matrices (Pérez et al., 2018; González et al., 2019; Llerena et al., 2023). In addition, nuclear magnetic resonance (NMR) is also frequently used for the overall metabolite analysis of cocoa beans and their fermentation products. NMR can simultaneously detect amino acids, organic acids, sugars and methyl xanthine, etc., providing metabolic fingerprints for cocoa beans of different varieties and origins (Caligiani et al., 2010; Boutchouang et al., 2024). Overall, LC-MS and GC-MS stand out more in terms of sensitivity and specificity, while NMR is more suitable for high-throughput, non-destructive testing and multi-component quantification. 3.2 Quantitative variation of theobromine across tissues, developmental stages, and cultivars The content of theobromine varies significantly among different tissues, developmental stages and varieties. Studies have found that theobromine levels are relatively high in young leaves and exocarp of fruits, gradually decreasing as tissues mature. However, in mature seeds, especially cotyledon, they accumulate in large quantities (Koyama et al., 2003; Zheng et al., 2004). There are also differences among different varieties. For example, the theobromine content in Nacional ×Trinitario type bean shells is higher than that in CCN-51 type (Llerena et al., 2023). In addition, the theobromine/caffeine ratio of Forastero and Trinitario varieties can be used as a chemical marker to distinguish varieties (Collazos-Escobar et al., 2024). Metabolomics studies have further revealed the dynamic distribution patterns of theobromine at different developmental stages and among tissues (Koyama et al., 2003; Zheng et al., 2004).
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